The role of backbone flexibility in the accommodation of variants that repack the core of T4 lysozyme.

To understand better how the packing of side chains within the core influences protein structure and stability, the crystal structures were determined for eight variants of T4 lysozyme, each of which contains three to five substitutions at adjacent interior sites. Concerted main-chain and side-chain displacements, with movements of helical segments as large as 0.8 angstrom, were observed. In contrast, the angular conformations of the mutated side chains tended to remain unchanged, with torsion angles within 20 degrees of those in the wild-type structure. These observations suggest that not only the rotation of side chains but also movements of the main chain must be considered in the evaluation of which amino acid sequences are compatible with a given protein fold

Thermodynamic and structural compensation in "size-switch" core repacking variants of bacteriophage T4 lysozyme.

Previous analysis of randomly generated multiple mutations within the core of bacteriophage T4 lysozyme suggested that the "large-to-small" substitution Leu121 to Ala (L121A) and the spatially adjacent "small-to-large" substitution Ala129 to Met (A129M) might be mutually compensating. To test this hypothesis, the individual variants L121A and A129M were generated, as well as the double "size-switch" mutant L121A/A129M. To make the interchange symmetrical, the combination of L121A with A129L to give L121A/A129L was also constructed. The single mutations were all destabilizing. Somewhat surprisingly, the small-to-large substitutions, which increase hydrophobic stabilization but can also introduce strain, were less deleterious than the large-to-small replacements. Both Ala129 --> Leu and Ala129 --> Met offset the destabilization of L121A by about 50%. Also, in contrast to typical Leu --> Ala core substitutions, which destabilize by 2 to 5 kcal/mol, Leu121 --> Ala slightly stabilized A129L and A129M. Crystal structure analysis showed that a combination of side-chain and backbone adjustments partially accommodated changes in side-chain volume, but only to a limited degree. For example, the cavity that was created by the Leu121 to Ala replacement actually became larger in L121A/A129L. The results demonstrate that the destabilization associated with a change in volume of one core residue can be specifically compensated by an offsetting volume change in an adjacent residue. It appears, however, that complete compensation is unlikely because it is difficult to reconstitute an equivalent set of interactions. The relatively slow evolution of core relative to surface residues appears, therefore, to be due to two factors. First, a mutation in a single core residue that results in a substantial change in size will normally lead to a significant loss in stability. Such mutations will presumably be selected against. Second, if a change in bulk does occur in a buried residue, it cannot normally be fully compensated by a mutation of an adjacent residue. Thus, the most probable response will tend to be reversion to the parent protein

Thermodynamic and structural compensation in "size-switch" core repacking variants of bacteriophage T4 lysozyme.

Previous analysis of randomly generated multiple mutations within the core of bacteriophage T4 lysozyme suggested that the "large-to-small" substitution Leu121 to Ala (L121A) and the spatially adjacent "small-to-large" substitution Ala129 to Met (A129M) might be mutually compensating. To test this hypothesis, the individual variants L121A and A129M were generated, as well as the double "size-switch" mutant L121A/A129M. To make the interchange symmetrical, the combination of L121A with A129L to give L121A/A129L was also constructed. The single mutations were all destabilizing. Somewhat surprisingly, the small-to-large substitutions, which increase hydrophobic stabilization but can also introduce strain, were less deleterious than the large-to-small replacements. Both Ala129 --> Leu and Ala129 --> Met offset the destabilization of L121A by about 50%. Also, in contrast to typical Leu --> Ala core substitutions, which destabilize by 2 to 5 kcal/mol, Leu121 --> Ala slightly stabilized A129L and A129M. Crystal structure analysis showed that a combination of side-chain and backbone adjustments partially accommodated changes in side-chain volume, but only to a limited degree. For example, the cavity that was created by the Leu121 to Ala replacement actually became larger in L121A/A129L. The results demonstrate that the destabilization associated with a change in volume of one core residue can be specifically compensated by an offsetting volume change in an adjacent residue. It appears, however, that complete compensation is unlikely because it is difficult to reconstitute an equivalent set of interactions. The relatively slow evolution of core relative to surface residues appears, therefore, to be due to two factors. First, a mutation in a single core residue that results in a substantial change in size will normally lead to a significant loss in stability. Such mutations will presumably be selected against. Second, if a change in bulk does occur in a buried residue, it cannot normally be fully compensated by a mutation of an adjacent residue. Thus, the most probable response will tend to be reversion to the parent protein

Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions.

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site

Mechanical unfolding of individual T4 lysozyme molecules

Mechanical unfolding of individual T4 lysozyme molecule

Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect.

Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins

Dissection of protein structure and folding by directed mutagenesis.

The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements. In order to determine the importance of 'alpha-helix propensity' in protein stability, different replacements have been made within alpha-helical segments of T4 lysozyme. Several such substitutions of the form Xaa-->Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favouring amino acid. It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity. This illustrates the redundancy that is present in the amino acid sequence. A number of 'cavity-creating' mutants of the form Leu-->Ala have been constructed to understand better the nature of hydrophobic stabilization. The structural consequences of these mutations differ from site to site. In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains. The destabilization of the protein associated with these cavity-creating mutations also varies from case to case. The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins

Dissection of protein structure and folding by directed mutagenesis.

The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements. In order to determine the importance of 'alpha-helix propensity' in protein stability, different replacements have been made within alpha-helical segments of T4 lysozyme. Several such substitutions of the form Xaa-->Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favouring amino acid. It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity. This illustrates the redundancy that is present in the amino acid sequence. A number of 'cavity-creating' mutants of the form Leu-->Ala have been constructed to understand better the nature of hydrophobic stabilization. The structural consequences of these mutations differ from site to site. In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains. The destabilization of the protein associated with these cavity-creating mutations also varies from case to case. The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins